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Wang et al. Cell Communication and Signaling 2014, 12:28http://www.biosignaling.com/content/12/1/28

RESEARCH Open Access

Autoinhibition of the Ron receptor tyrosine kinaseby the juxtamembrane domainXin Wang1, Neela Yennawar2 and Pamela A Hankey1,3*

Abstract

Background: The Ron receptor tyrosine kinase (RTK) has been implicated in the progression of a number ofcarcinomas, thus understanding the regulatory mechanisms governing its activity is of potential therapeuticsignificance. A critical role for the juxtamembrane domain in regulating RTK activity is emerging, however themechanism by which this regulation occurs varies considerably from receptor to receptor.

Results: Unlike other RTKs described to date, tyrosines in the juxtamembrane domain of Ron are inconsequentialfor receptor activation. Rather, we have identified an acidic region in the juxtamembrane domain of Ron that playsa central role in promoting receptor autoinhibition. Furthermore, our studies demonstrate that phosphorylation ofY1198 in the kinase domain promotes Ron activation, likely by relieving the inhibitory constraints imposed by thejuxtamembrane domain.

Conclusions: Taken together, our experimental data and molecular modeling provide a better understanding ofthe mechanisms governing Ron activation, which will lay the groundwork for the development of noveltherapeutic approaches for targeting Ron in human malignancies.

Keywords: Ron, Receptor tyrosine kinase, Autoinhibition, Juxtamembrane domain

BackgroundDysregulated RTK activity is closely associated with abroad range of human malignancies. Thereby, under-standing the complex mechanisms by which RTKs areregulated is of clinical significance due to potential ap-plications for therapeutic intervention against cancers[1,2]. Overexpression or constitutive activation of theRon receptor has been demonstrated to drive strongoncogenic phenotypes in cancers derived from breast,colon, lung, and prostate [3-5]. The oncogenic potentialof Ron is represented by its capacity to induce migration,invasion, growth, survival and epithelial-mesenchamaltransition of epithelial tumor cells, as well as to promotepro-tumoral activities of tumor-associated macrophages[6-13]. Inhibition of Ron expression using Ron specificsiRNA dramatically decreases tumorigenic and invasiveactivities in colorectal carcinoma cells [14]. A human

* Correspondence: [email protected] Program in Cell and Developmental Biology, The PennsylvaniaState University, University Park, PA 16802, USA3Department of Veterinary and Biomedical Sciences, The Pennsylvania StateUniversity, 115 Henning Building, University Park, PA 16802-3500, USAFull list of author information is available at the end of the article

© 2014 Wang et al.; licensee BioMed Central LCommons Attribution License (http://creativecreproduction in any medium, provided the or

neutralizing antibody against Ron exhibits partial inhib-ition of colon, lung and pancreatic tumor growth in xeno-graft models [15], and a small molecule kinase inhibitor ofRon that has antitumor activity in vivo has been described[16]. Consequently, Ron is a promising target for the-rapeutic intervention against tumorigenic activities andmalignant phenotypes [3,17,18].Recepteur d'originenantais (Ron) (called STK in mice

and Sea in chickens) belongs to the Met proto-oncogenefamily [19,20]. Originally synthesized as a single chain pre-cursor, Ron is processed into a disulfide-linked heterodi-mer with a transmembrane β chain and an extracellular αchain [21,22]. Binding of macrophage stimulating protein(MSP), the ligand for Ron, to the extracellular domain ofRon induces receptor dimerization, conferring catalyticactivity to the receptor [23]. The intracellular region ofRon includes the juxtamembrane domain, the highlyconserved kinase domain and the non-catalytic c-terminaltail. The kinase domain consists of two lobes. The N-lobecontains the αC helix and the P loop which are essentialfor ATP recruitment, while phosphorylation of two tyro-sines (Y1238 and Y1239) in the C-lobe activation loop is re-quired for receptor activation. Subsequent phosphorylation

td. This is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andiginal work is properly credited.

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Wang et al. Cell Communication and Signaling 2014, 12:28 of 10http://www.biosignaling.com/content/12/1/28

of two docking tyrosines in the c-terminal tail transduces avariety of signaling pathways mediated by Ron [22,24](Figure 1A).The juxtamembrane domain, which forms an import-

ant layer of constraint, limiting promiscuous enzymaticactivity of RTKs, is found frequently mutated in tumorcells [25-32]. The regulatory mechanisms mediated bythe juxtamembrane domain however, varies significantlydue to the varied length and lack of sequence similarityamong RTKs. Murine Ron harboring a deletion of 27residues in the juxtamembrane domain, caused by exclu-sion of a potential exon, exhibits significantly higherreceptor activity than human Ron [33-36]. Deletion ofthe 27 residues in human Ron promotes receptor activa-tion, highlighting a potential role for the juxtamembranedomain in the regulation of the Ron receptor activation[33]. However, the molecular mechanisms underlyingthis regulation are unknown.Herein, we have characterized the mechanism by

which the juxtamembrane domain contributes to theauto-regulation of Ron activity through structure/functionanalysis. While tyrosines in the Ron juxtamembranedomain are inconsequential for receptor activation, anacidic JM-C region in the juxtamembrane domain wasfound to be critical for Ron autoinhibition. Phosphoryl-ation of Y1198 in the kinase domain promoted Ron activa-tion, primarily by accelerating relief of the autoinhibition

Figure 1 Schematic diagram of the Ron receptor. A) Domains of the Roand activation loop (yellow) in the C lobe are denoted by different colors.tail (P-Y: Y1353,1360) are indicated. B) The juxtamembrane domain of Ron is dregion at the N terminus; JM-B (1009–1035), the region corresponding to thepatch of residues; and JM-D (1048–1081), the region at the c-terminus of the

imposed by the JM-C region. Based on our experimentaldata, we have proposed molecular models of the inactiveand the active Ron structure, which cast a new lighton the mechanisms controlling Ron mediated signaltransduction.

ResultsThe acidic JM-C region in the Ron juxtamembrane domainplays a critical role in receptor autoinhibitionThe juxtamembrane domain of Ron is significantlylonger than that of most RTKs, therefore we divide itinto four sequential regions for clarity (Figure 1B). Pre-vious studies have mapped the functional differencebetween murine Ron and human Ron to the 27 residuescomprising the JM-B region, which contains Y1012 andY1017, the only two tyrosines in the juxtamembranedomain (Additional file 1: Figure S1A). Tyrosines in thejuxtamembrane domain of RTKs such as the EphR, Flt3and Kit [29,32,37] are critical for maintenance of theinactive kinase, and phosphorylation of these tyrosinesrelieves receptor autoinhibition. However, consistentwith previous reports [33,38], mutation of Y1012 and Y1017

individually or in combination into phenylalanine, glu-tamic acid or alanine in the context of Ron did not affectreceptor autophosphorylation, downstream Erk phosphor-ylation (Additional file 1: Figure S1B), or induction of AP1luciferase activity (Additional file 1: Figure S1C). Thus,

n receptor. P loop (orange) and αC helix (blue) in the kinase N lobeCritical tyrosines in the activation loop (P-2Y: Y1238, 1239) and c-terminalivided into four sequential regions: JM-A (residue 982–1008), thesequence deleted in mRon; JM-C (1036–1047), the region with an acidicjuxtamembrane domain. Residues discussed herein are indicated.


unlike other RTKs, tyrosines in the juxtamembrane do-main of Ron do not regulate receptor activation.While the primary sequence differs, acidic residues

(glutamic acid and aspartic acid) interspersed with ser-ines in the Ron JM-C region (Figure 2A) are relativelyconserved in the closely related Met receptor, suggestingthat this region might serve a conserved function. Inorder to determine the potential role of this region inthe regulation of Ron receptor activity, we constructed aRon mutant in which nine residues (most acidic) in theJM-C region were deleted (RonΔ1039–1047) (Figure 2A).HA-tagged wild-type Ron and Ron variants were transi-ently transfected in HEK 293 cells. Phosphorylation ofthe activation loop tyrosines (Y1238/9), a hallmark ofkinase activation, and induction of a major signalingcascade downstream of Ron, the Ras-Map kinase pathway,

Figure 2 Acidic residues in the JM-C region of Ron promotereceptor autoinhibition. A) Schematic of mutants. Mutations arehighlighted in bold. Residues deleted are underlined. B) 293 cellswere transfected with HA-tagged wild-type or mutant Ron asindicated in the presence and absence of MSP. Lysates were blottedfor p-Ron1238/9Y, HA, p-Erk, Erk and β-tubulin. C) Relative luciferaseactivity in 293 cells co-transfected with wild-type or mutant Ron asindicated, and an AP-1 luciferase reporter in the presence andabsence of MSP. The data were presented as the average ± standarddeviation from measurements collected from three independentexperiments and three measurements per experiment. Wild typeRon −/+MSP was used as controls, unless denoted with ‘︹’. Thesignificant differences of values at p < 0.05, p < 0.01, and p < 0.001levels are indicated by *, **and ***respectively.

were examined. Ron and the variants were also transientlytransfected with an AP-1 luciferase reporter, and theinduced AP-1 transcriptional response was measured.Equivalent levels of protein expression and cell surfaceexpression of Ron and its variants were demonstratedby probing for HA and by the flow cytometry analysis(Additional file 2: Figure S2), respectively. Comparedwith the wild type receptor, RonΔ1039–1047 exhibited asignificant increase in receptor autophosphorylation,induced Erk phosphorylation (Figure 2B) and AP-1transcriptional activity both in the presence and absenceof MSP (Figure 2C). In order to further determinewhether the negative electrostatic nature of the JM-Cregion is responsible for its role in the regulation of Ronactivity, E1044, D1045 and E1046 in the JM-C region weremutated to alanine individually and in combination. Singlemutants did not show remarkable catalytic differencescompared with wild type Ron (data not shown). However,the triple mutation (EDE-AAA) dramatically upregulatedreceptor activity and downstream signaling to a level com-parable to that induced by RonΔ1039–1047 (Figure 2B and C).Based on these observations, we have identified a novelsegment in the juxtamembrane domain of Ron, the JM-Cregion, which plays a central role in kinase autoinhibition,primarily mediated by the acidic residues.Serines (S1039,S1041,S1043,S1047) are major components

of the acidic JM-C region, the first three of which arepredicted to be potential phosphorylation sites (NetPhos2.0 Server) (data not shown) (Figure 3A). In order todetermine whether phosphorylation of these serinescould play a role in the regulation of Ron receptor activityby the JM-C region, we mutated the serines to alaninesindividually and in combination. While individual anddouble mutants performed similarly as wild-type Ron(data not shown), the triple and quadruple mutations (3S-A and 4S-A) resulted in an evident increase in receptoractivation and downstream signaling (Figure 3B and C).However, this activation was not as striking as that drivenby mutations of the acidic 1044EDE residues. Thus, whilethe acidic residues in the JM-C region maintain the pri-mary inhibitory constraints, phosphorylation of serines inthis region may also contribute to the Ron autoinhibitionmediated by the JM-C region.

The JM-B region regulates receptor activity indirectlythrough its impact on the spatial orientation of the JM-CconstraintPrevious studies demonstrated that deletion of the JM-Bregion of human Ron (P1009-V1035), absent in murineRon, resulted in elevated receptor activity, as observedin murine Ron [33]. However, our studies suggest thatindividual residues in the JM-B region including tyro-sines, are dispensable for Ron activation. Therefore, wereasoned that the JM-B region might play a structural

Figure 3 Serines in the JM-C region play a subsidiary role inthe regulation of Ron receptor activation. A) Schematic ofmutants. Mutations are highlighted by underline. B) 293 cells weretransfected with HA-tagged wild-type or mutant Ron as indicatedin the presence and absence of MSP. Lysates were blotted forp-Ron1238/9Y, HA, p-Erk and Erk. C) Relative luciferase activity in 293cells co-transfected with wild-type or mutant Ron as indicated, andan AP-1 luciferase reporter in the presence and absence of MSP.


role in Ron autoinhibition. Consistent with previousstudies, here we found that RonΔJM-B possessed higherintrinsic catalytic capability than the wild type receptor,as demonstrated by a dramatic increase in phosphoryl-ation of the activation loop tyrosines and a docking tyro-sine, and in expression of an AP-1 reporter in both thepresence and the absence of ligand (Figure 4B and C). Inorder to determine the significance of the length of theJM-B region, a Ron variant with a smaller deletion of tenresidues (G1020-L1029) in the JM-B region, RonΔJM-B-S,which did not result in deletion of the tyrosines in theJM-B region, was constructed (Figure 4A). Surprisingly,while RonΔJM-B exhibited enhanced receptor activity,RonΔJM-B-S exhibited decreased receptor activity, and theinduced AP-1 transactivation was significantly reducedeven in the presence of MSP (Figure 4C and D). Thesedata indicate that the length of the JM-B region, ratherthan individual residues, appears to be a critical deter-minant for the proper regulation of Ron activity.Since the acidic JM-C region lies c-terminal to the

JM-B region of Ron, we hypothesized that the JM-Bregion might modulate receptor activity indirectly byaffecting localization of the JM-C region. In order to

test this hypothesis, the EDE-AAA mutation in theJM-C region was generated in the context of RonΔJM-B

and RonΔJM-B-S (Figure 4A). Since both ΔJM-B and EDE-AAA were able to relieve Ron autoinhibition, as expected,RonΔJM-B&EDE-AAA exhibited enhanced catalytic activityboth in the presence and absence of MSP (Figure 4Dand E). However, the levels of AP-1 luciferase activityinduced by RonΔJM-B&EDE-AAA were similar to thoseinduced by RonEDE-AAA, suggesting that the deletion ofJM-B region and neutralization of the acidic JM-C re-gion may facilitate receptor activation in an analogousmanner. Alternatively, although the ΔJM-B-S deletionprohibited receptor activation, introduction of the EDE-AAA mutation into the RonΔJM-B-S construct largely re-stored receptor activity (Figure 4D and E). Thus, it is likelythat the JM-B region modulates activation of the Ron re-ceptor by affecting the spatial orientation of the adjacentJM-C region, which places autoinhibitory constraints onthe kinase due to its electronegative characteristics.

Y1198 in the kinase domain mediates Ron autoinhibitionimposed by the JM-C regionPrevious studies indicate that Y1194 in Met, the equi-valent residue of Y1198 in Ron, is one of the three majorreceptor autophosphorylation sites, and mutation ofY1194 to phenylalanine in Met results in dramaticallydecreased receptor activity [39,40]. Here we show thatthe equivalent Y1198F mutation in Ron dramaticallyreduced Ron autophosphorylation, as well as inductionof downstream Erk phosphorylation and AP-1 transcrip-tional activation both in the presence and absence ofMSP (Figure 5B and C). These results indicate thatphosphorylation of Y1198 in the αE helix of the kinase Clobe plays a key role in the activation of wild type Ron.Although Y1198 and the juxtamembrane domain areunlikely to sterically interact with each other based ontheir distance, the potential functional interrelation wasevaluated by introduction of the Y1198F mutation in thecontext of RonEDE-AAA and RonΔ1039–1047 (Figure 5A).Alterations in the JM-C region, which were demonstratedto relieve Ron autoinhibition, successfully rescued the lossof receptor activity caused by the 1198Y-F mutation. Thedouble mutants recovered receptor activity to a level com-parable to wild type Ron, and responded to stimulation byMSP (Figure 5D and E). These studies suggest that phos-phorylation of Y1198 may play an essential role in relievingthe inhibitory constraints on Ron activation imposed bythe JM-C region in an indirect manner.

DiscussionMutations in the juxtamembrane domain are a commonmechanism by which RTKs become constitutively acti-vated and drive progression of a variety of human cancers.Internal tandem duplications in the juxtamembrane

C

B

A

D

E

Figure 4 The JM-B region modulates Ron receptor activation by affecting the orientation of the JM-C region. A) Schematic of mutants.Deletions of different lengths in the JM-B region are indicated by arrows. Mutated residues are highlighted in bold. B) & D) 293 cells weretransfected with HA-tagged wild-type or mutant Ron in the presence and absence of MSP. Lysates were immunoprecipitated with anti p-Y andblotted for HA, or directly blotted for p-Ron1238/9, p-Ron1360 and HA, p-Erk and Erk. C) & E) Relative luciferase activity in 293 cells co-transfectedwith wild-type or mutant Ron, and an AP-1 luciferase reporter in the presence and absence of MSP.


domain of Flt3 have been identified in patients with acutemyeloid leukemia [41]. Deletions in the juxtamembranedomain of both KIT [42] and PDGFR [28] have been iden-tified in human gastrointestinal stromal tumors. Germ linepoint mutations in the juxtamembrane domain of Methave been identified in small cell lung cancer [27] andgastric cancer [43]. An alternatively spliced form of Met,missing exon 14 which encodes part of its juxtamembranedomain [44], exhibits a highly tumorigenic phenotype[31,45]. Likewise, a splice variant of RON, missing exon14 (BG289902) in the juxtamembrane domain, was foundin a bladder papilloma cell line (human ExpressedSequence Tag data base). The juxtamembrane domainof RTKs varies in length and lacks apparent sequencesimilarity, suggesting that it may contribute to specificaspects of regulation within individual receptor families.Our studies demonstrate that, like other RTKs, the juxta-membrane domain of Ron plays a central role in receptorautoinhibition. However, the mode of regulation conferredby the juxtamembrane domain of Ron appears to be novelcompared with other RTKs described to date.Tyrosine phosphorylation in the juxtamembrane

domain of RTKs including Kit, Flt3 and EphR, is a

common mechanism by which the receptor switchesfrom the default closed state to an active configuration[29,32,37,46]. However, phosphorylation and the aro-matic side chain of tyrosines in the JM-B region aredispensable for Ron activity. Conversely, deletions ofdifferent lengths in the JM-B region were found to havesignificant but opposite impacts on receptor activation,probably through affecting the spatial orientation of thecontiguous JM-C region.Here, we show that the acidic cluster in the JM-C re-

gion of Ron is a crucial regulator of receptor activity,and disruption of its negative electrostatic property dra-matically facilitates kinase activation. The acidic charac-teristics of the JM-C region are relatively conservedwithin the Met family members. The equivalent regionin Met was found to contain a mutation (S1058P) in asample from the non small cell lung cancer [47], sug-gesting the potential functional significance of thisregion. In order to better understand the inhibitorymechanism imposed by the JM-C region, we comparedthe kinase domain sequences of 150 receptors by mappingthem onto the crystal structure of the Ron kinase domain(3PLS) [48] (Figure 6). Based on surface accessibility and

B C

AD

E

Figure 5 Y1198 in the kinase domain of Ron regulates the autoinhibitory constraints on receptor activation imposed by the JM-Cregion. A) Schematic of the Ron receptor. Y1198 at the interface of the αE helix and the αC helix is highlighted. B) & D) 293 cells were transfectedwith HA-tagged wild-type or mutant Ron as indicated in the presence and absence of MSP. Lysates were blotted for p-Ron1238/9Y, HA, p-Erkand Erk. C) & E) Relative luciferase activity in 293 cells co-transfected with wild-type or mutant Ron as indicated, and an AP-1 luciferase reporter inthe presence and absence of MSP.


relative location, a conserved region on the surface of theRon kinase N lobe was identified as a putative JM-C bind-ing region (orange in Figure 6A). It locates adjacent to thestrictly conserved active site (red in Figure 6A), with a pre-dominantly positive surface potential (blue in Figure 6B).Based on the crystal structure of the Ron kinase

domain, we built a molecular model of the inactive Ronstructure (including JM-C, JM-D region and kinasedomain) [48-51] (Additional file 3: Figure S3A), whichsuggests that the JM-C region (magenta) promoting ahelical conformation could make close contacts with theP loop (orange) and activation loop (yellow). Acidicresidues in the JM-C region (E1044-D1045-E1046) have thepotential to interact with H1092 in the P-loop, Y1238 andQ1243 in the activation loop, and the side-chains of S1117

and R1118 between the β3 strand and the αC helix (linesin Additional file 3: Figure S3A). These interactionswould interfere with the accommodation of ATP andsubstrates by the glycine rich P-loop. Spatial proximity

of the JM-C region could also lock the activation loop inthe inter-lobe cleft and prevent productive rotation ofthe N and C lobe. In addition, the rigid configurationpromoted by the JM-C region, of the β3 strand and αChelix in the kinase N lobe, puts the invariant K1114 andE1130 that participate in formation of a salt bridge re-quired for ATP binding in catalytically disfavoredpositions.Located at the interface between the αE and the αC

helix, Y1198 of Ron is highly conserved among RTKs andshares structural conservation among close relatives in-cluding Met, Ret, the insulin receptor, IGF1R and FGFR[40,52-55]. Phosphorylation of the equivalent tyrosine inMet and murine Ron is closely associated with receptoractivity [36,39,40]. In the crystal structure of the Ronkinase domain [48], the nonproductive orientation of theαC helix is held by two hydrogen bonds (Additional file 4:Figure S4). One is between the side chain hydroxyl ofY1198 in the αE helix and the backbone amide of N1139 at

Figure 6 Conserved surface residues and surface potential ofthe Ron kinase domain (3PLS) [48]. The conserved regionadjacent to the active site with a predominantly positive surfacepotential is indicated by arrows as the putative JM-C binding site.A) Conserved residues on the surface of human kinase domains areindicated, with red/orange representing highly conserved residuesand blue/cyan representing highly variable residues. B) Surfacepotential of the Ron kinase domain, with blue representing positivepotential and red representing negative potential. The units of thescale were −60.362 ~ 60.362.


the C-terminus of the αC helix, and the other one is be-tween R1231 and Q1124 at the N-terminus. In order to fur-ther understand the events leading to receptor activation,we modeled the activated Ron kinase domain based onthe crystal structure of active Met (3Q6U) [56] (Additionalfile 3: Figure S3B). In this model, the hydrogen bondbetween Y1198 and N1139 is disrupted, potentially due tothe phosphorylation of Y1198. Consequently, the regionalstability of the inactive kinase domain collapses, and theαC helix reorients significantly compared to its position inthe auto-inhibited model. This large scale movementcould promote the displacement of the JM-C constraint,resulting in reorientation of the P-loop and activation

loop, and realignment of the active site residues for re-cruitment of ATP and substrates. Therefore, when Y1198 ismutated to phenylalanine, the absence of phosphorylationat this residue would be predicted to fix the αC helix inthe kinked non-productive orientation, stabilizing thekinase in the inactive state. However, when the inhibitoryJM-C region is released by alterations in the juxtamem-brane domain that disrupt the autoinhibition confered bythe juxtamembrane domain, phosphorylation of Y1198

would not be required for reorientation of the kinase seg-ments and receptor activation.

ConclusionsBecause the mode of regulation by the juxtamembranedomain varies significantly among RTKs, targeting thistier of regulation could provide an opportunity to de-velop inhibitors that would exploit the unique aspects ofjuxtamembrane domain regulation conferred upon indi-vidual receptors. In that context, a synthesized peptiderepresenting the juxtamembrane domain of Kit is able toimpede receptor activation in vitro and delay growth ofcells containing an oncogenic form of Kit [37]. Alterna-tively, targeting the autoinhibited form of the receptorwith small molecule inhibitors could provide an add-itional level of specificity due to the association betweenthe juxtamembrane domain and the active site. Thework presented here has unveiled a novel regulatorymechanism governing Ron activity by its juxtamembranedomain, and provided a new perspective regarding theregulation of RTK activity through conformational plas-ticity and functional cooperation of different protein seg-ments, which may provide important insights that couldlead to the development of a novel class of therapeuticsfor the treatment against a wide range of Ron mediatedcarcinomas.

MethodsGene construction and mutagenesisHuman RON and mutants were expressed by the mam-malian pcDNA3.1 vector or the murine stem cell virus(MSCV) retroviral vector. Mutants were generated frompcDNA-RON-HA or MSCV-RON-HA, with the QuikChange mutagenesis kit (Stratagene) according to themanufacturer’s instructions. The following PCR protocolwas used and primers for the mutants are listed inAdditional file 5: Table S1: 95°C 30 s, followed by 95°C30 s, 55°C 1 min, 68°C 12 min for 20 cycles, and a final68°C 12 min.

Cell culture, antibodies, and reagentsHuman embryonic kidney (HEK) 293 cells (ATCC) weremaintained in Dulbecco’s modified Eagle’s medium sup-plemented with 10% fetal bovine serum. The TransIT-293 transfection reagents were purchased from Mirus


Bio, LLC (Madison, WI). The dual-luciferase reporterassay system was purchased from Promega Corporation(Madison, WI). Antibodies against Ron 1238/9phospho-tyrosine, phospho-Erk, Erk and the HA epitope (262 K)were purchased from Cell Signaling. Goat anti-rabbit IgG-HRP was purchased from Santa Cruz Biotechnology, Inc(Santa Cruz, CA). Recombinant MSP was purchased fromR&D Systems. The AP-1 luciferase reporter cDNA waskindly provided by Dr. Avery August (Cornell University).All PCR primers were ordered from Eurofins MWGOperon, Inc. (Huntsville, AL). Pfu Turbo DNA polymer-ase was purchased from Agilent Tech. ECL Plus Westernblotting detection reagents were purchased from GEHealthcare (Piscataway, NJ).

Cell transfection and luciferase assaysFor the luciferase assay, 5 × 104 HEK 293 cells/well wereseeded into 24-well plates. Twenty four hours later, adesignated mixture of 60 ng of wild type or mutantforms of RON, 60 ng of AP-1 luciferase reporter plas-mid, and 0.5 ng renilla were used for transient transfec-tion with the Mirus-293 transfection reagent accordingto the manufacturer’s protocol. MSCV-neo or PCDNA3.1-neo plasmid were used as control vectors for each trans-fection. Cells were stimulated with 50 ng/ml (50 ug/mlstock) MSP 24 h following transfection. Luciferase assayswere performed at 24 h later according to the manufac-turer’s instructions (Promega). The fold increase is calcu-lated as the firefly luciferase activity divided by the renillaluciferase activity.

Western blot analysisA total of 2.5 × 105HEK 293 cells/well were plated intosix-well plates. Twenty four hours later, the cells weretransiently transfected with 300 ng RON or its deriva-tives per well. Cells were stimulated with 200 ng/mlMSP for 48 h following transfection. 10 minutes later,cells were suspended in 400 ul ice-cold cell lysis buffercontaining 150 mM NaCl, 20 mM Tris–HCl (pH 7.5),5 mM EDTA, 1% NP-40, 1 mM phenylmethylsulfonylfluoride, 1 mM Na3VO4, and 10 mM NaF. Cell lysateswere centrifuged at 12,000 rpm for 20 min, and thesupernatant lysates were transferred to prechilled tubes.Cell lysates were mixed with 4X denaturing SDS loadingbuffer and heated at 100°C for 8 min. Samples were re-solved on an SDS-PAGE gel and then transferred topolyvinylidene difluoride membranes. Membranes werethen blocked with 5% nonfat milk or Bovine SerumAlbumin in Tris-buffered saline with 0.1% Tween 20(TBST) for 1 h and probed with primary antibody at 4°Covernight. Membranes were washed three times in TBSTand incubated with secondary goat anti-rabbit IgG-HRPfor another hour. Membranes were washed three timesin TBST before ECL plus Western blotting detection

reagents were applied for visualization. For reprobing,membranes were stripped with 62.5 mM Tris–HCl(pH 6.8), 2% SDS, and 0.7% β-mercaptoethanol at 55°Cfor 30 min.

Flow cytometryA total of 1.2 × 105 HEK 293 cells/well were plated into12-well plate. Twenty four hours later, the cells weretransiently transfected with 200 ng RON or its deri-vatives per dish. PCDNA3.1-neo plasmid was used ascontrol vectors for each transfection. Flow cytometryanalysis was performed at 48 h later. 106 cells were incu-bated per tube in 50 ul FACS buffer (ice cold PBS + 2%FBS). Non-specific binding was blocked by the additionof 50 μL HAB for 2 minutes at room temperature. Cellswere incubated on ice with 5 ug/ml anti-Ronβ (DX07)or IgG (control) for 30 min. Cells were washed twicewith 1 ml FACS buffer and resuspended once more in50 μL of FACS buffer. 50 μL HAB per tube was addedagain for blocking of non-specific binding. Cells wereincubated with 5 ug/ml PE anti-mouse IgG1 as a second-ary antibody to Ron for 30 min on ice. Cells were washedtwice, resuspended in 1 mL of FACS buffer and analyzedfor FLOW on a Beckman-Coulter FC500.

Evaluation of surface conservation and potentialA comparison of 150 receptor kinase domain sequenceswas mapped onto the crystal structure of the Ronkinase domain (PDB code 3pls) using the CONSURFweb server. Surface conservation and surface potential ofthe Ron kinase domain were illustrated. Potential JM-Cbinding site was proposed based on residue/sequencecharacteristics and relative distance.

Molecular modeling of the JM-C regionModeling of the autoinhibited and the activated con-formation of Ron was based on the crystal structure ofthe kinase domains of Ron (PDB code 3PLS) and Met(PDB code 3Q6U) respectively. The modeling was donemanually using the COOT software [49]. Hydrogenbonds between side chains were manually optimized andthe resultant model was energy minimized by using theYasara server [51].

Structure-based sequence analysisSeven algorithms, BPS [16], D_R [57], DSC [58], GGR[59], GOR [60], G_G [61], H_K [62], K_S [63], L_G [64],and Q_S [65], were used for secondary structure predic-tion. The Joint prediction (JOI) is the prediction madeby the program that assigns the structure using a “win-ner takes all” approach.


Additional files

Additional file 1: Figure S1. Tyrosines in the juxtamembranedomain of Ron are dispensable for the regulation of receptor activity.A) Schematic diagram of mutants. Y1012 and Y1017 in the JM-B region arehighlighted in bold. B) 293 cells were transiently transfected with HA-tagged wild-type or mutant Ron in the presence and absence of MSP.Lysates were blotted for p-Ron1238/9, HA, p-Erk and total Erk. C) Relativeluciferase activity in 293 cells co-transfected with wild-type or mutantRon, and an AP-1 luciferase reporter in the presence and absence of MSP.

Additional file 2: Figure S2. Cell surface expression of wild type Ronand the Ron variants discussed herein. 293 cells were transientlytransfected with wild-type or mutated Ron as indicated. Cells wereharvested 48 h after transfection and analyzed by flow cytometry forprotein membrane expression.

Additional file 3: Figure S3. Predicted structure of the autoinhibitedand active form of Ron. A) In the inactive Ron model, the JM-C region(magenta) is predicted to pack against and interact with the activationloop (yellow) and P-loop (orange) near the active site of the kinasedomain. Potential bonds among residues in the JM-C region (1044EDE)(magenta), the P loop (H1092) (orange), the active site (S1117, R1118) (green)and the activation loop (E1237, Y1238) (yellow) are labeled with blackdashed lines. Y1198 (cyan stick) in the kinase C-lobe hydrogen bonds tothe backbone amide of N1139 at the c-terminus of the αC helix. B) In theactive Ron model, phosphorylation of Y1198 results in reorientation of theαC helix and JM-D region. Consequently, the JM-C region is released fromthe active site, which enables ATP binding and reorientation of theactivation loop. The orange and cyan stick represents AMP-PNP.Figure was generated using the software Pymol.

Additional file 4: Figure S4. Y1198-N1139 hydrogen bond promotes thenon productive orientation of the αC helix. Crystal structure of theautoinhibited Ron kinase domain (3PLS) is shown as cartoon. The αChelix and αE helix are colored in cyan and green respectively. Residuesinvolved in the hydrogen bonds (yellow dash) linking the two helices arelabeled in white.

Additional file 5: Table S1. PCR primers for the mutant construction.

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsXW performed all the experiments and wrote the manuscript, NY performedall of the molecular modeling, PAH designed the experiments and helpedwrite the manuscript. All authors read and approved the final manuscript.

Author details1Graduate Program in Cell and Developmental Biology, The PennsylvaniaState University, University Park, PA 16802, USA. 2Huck Institutes for LifeSciences, The Pennsylvania State University, University Park, PA 16802, USA.3Department of Veterinary and Biomedical Sciences, The Pennsylvania StateUniversity, 115 Henning Building, University Park, PA 16802-3500, USA.

Received: 9 July 2013 Accepted: 5 February 2014Published: 16 April 2014

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doi:10.1186/1478-811X-12-28Cite this article as: Wang et al.: Autoinhibition of the Ron receptortyrosine kinase by the juxtamembrane domain. Cell Communication andSignaling 2014 12:28.

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